The Journal of Basic & Applied Zoology (2012) 65, 184–190 The Egyptian German Society for Zoology The Journal of Basic & Applied Zoology www.egsz.org www.sciencedirect.com Phylogenetic relationship among five geckos from Egypt based on RAPD-PCR and protein electrophoresis (SDS–PAGE) Nadia H.M. Sayed Zoology Dept., College for Women for Science, Arts and Education, Ain Shams University, Heliopolis, Cairo, Egypt Received 28 February 2012; accepted 6 March 2012 Available online 24 September 2012 KEYWORDS Abstract Genetic variations between five gekkonid species from Egypt; Tropiocolotes tripolitanus, Gekkonidae; Tropiocolotes steudneri, Tropiocolotes nattereri, Tarentola mauritanica and Tarentola annularis were Protein electrophoresis; analyzed by SDS–PAGE for water soluble proteins and random amplified polymorphic DNA RAPD-PCR; (RAPD) analysis. Based on SDS–PAGE of water soluble proteins for all species, the obtained Phylogenetic relationship; results revealed a total of 17 bands at molecular weights that ranged from 95 to 16 kDa. The poly- Geckos morphic bands among species were 11 (64.7%) and the mean similarity matrix value between them was 70.7%. Using RAPD-PCR, the results showed eight total amplified bands at molecular weights that ranged from 1408 to 360 bp. The polymorphic bands between species were 7 (87.5%) and the mean similarity matrix between them was 44.6%. The dendrogram showed that, the five gekkonid species are separated from each other into two clusters. The first cluster contains three species of the genus Tropiocolotes. The second cluster includes the two species of the genus Tarentola. Based on SDS–PAGE and RAPD-PCR results, T. nattereri is sister to T. steudneri with higher genetic sim- ilarity than with T. tripolitanus. It is concluded that, the similarity coefficient and the genetic dis- tance values between the five gekkonid species indicate that the five gekkonid species are not identical and are separated from each other. From these results, it is indicated that the protein and RAPD analysis are useful molecular tools to indicate genetic variation between the species in the same genus or in the different genera. ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved. Introduction The squamates are the most diversified group containing liz- E-mail address: [email protected] ards and snakes (Vidal and Hedges, 2009). Gekkonidae is Peer review under responsibility of The Egyptian German Society for one of the largest vertebrate group among squamates that Zoology. are distributed throughout the world (Vidal and Hedges, 2005). There are four subfamilies of the Gekkonidae (Diplo- dactylinae, Gekkoninae, Eublepharinae and Sphaerodactyli- Production and hosting by Elsevier nae) with 1130 species and 108 genera (Han et al., 2004). 2090-9896 ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jobaz.2012.07.004 Phylogenetic relationship among five geckos from Egypt based on RAPD-PCR and protein electrophoresis (SDS–PAGE) 185 Geckos have high diversity in arid and semiarid habitats in Africa and Australia (Baha El Din, 2006). Several investiga- tions have been recorded on the herpetofauna of reptiles in Egypt (Anderson, 1898; Marx, 1968; Goodman and Hobbs, 1994; Baha El Din, 2006). The phylogenic relationship among the species of the family Gekkonidae was based on morpho- logical (Anderson, 1898; Marx, 1968), karyological (Kawai et al., 2009) and molecular studies (Joger, 1985; Rato et al., 2010; Busais and Joger, 2011). The genus Tarentola comprises about 20 morphologically similar species (Baha El Din, 1997; Harris et al., 2004a,b), which live mainly in semi-arid to arid habitats. They are dis- tributed in North Africa, coastal regions of Mediterranean Sea, Macaronesian islands and also Cuba and the Bahamas. Tarentola mauritanica is distributed in Mediterranean regions (North Egypt) and extends across Northern Libya and South- ern Tunisia. The Tarentola annularis is distributed in a wide area across Africa, central Sudan, to the north along the Nile to the Nile Delta and Sinai Peninsula (Egypt) (Joger, 1984a; Figure 1 Gel electrophoresis represents protein bands from five Baha El Din, 2006). The phylogenetic studies of the genus Gekkonid species (lanes 1–5). M, protein marker with molecular Tarentola based on molecular analysis such as serum protein size (1 kDa). 1, Tropiocolotes steudneri;2,Tropiocolotes nattereri; electrophoresis, quantitative precipitin tests of serum albumin 3, Tropiocolotes tripolitanus;4,Tarentola mauritanica;5,Tarentola (Joger, 1984b, 1985), RAPD-PCR analysis (Ali, 2012), DNA annularis. sequences from mitochondrial and nuclear genes (Carranza et al., 2002; Jesus et al., 2002; Harris et al., 2004a,b; Perera and Harris, 2008) indicated that morphologically similar spe- Materials and methods cies exhibit genetic variation. The genus Tropiocolotes (Peters, 1880) comprises a group of Five gekkonid species from Egypt were collected from differ- small, nocturnal and ground dwelling geckos. Several authors ent localities (Table 1). The five species are belonging to two (Baha El Din, 2001; Rastegar-Pouyani et al., 2007; Wilms genera. Morphological identification and classification of ani- et al., 2010) studied the morphological characters of Tropiocol- mals as well as scientific and common names of these species otes steudneri, Tropiocolotes nattereri and Tropiocolotes tripo- were carried out according to previous works (Anderson, litanus. The distribution of Tropiocolotes ranges from the 1898; Marx, 1968; Goodman and Hobbs, 1994). Western Sahara across Northern Africa to Israel, Sinai, the Arabian Peninsula, Iran, eastern Afghanistan and Pakistan (Anderson, 1898; Rastegar-Pouyani et al., 2007). Molecular Protein electrophoresis (SDS–PAGE) studies on the genus Tropiocolotes (Ali, 2012) are very scarce. Molecular markers proven by analysis of proteins and Protein electrophoresis was carried out with sodium dodecyl RAPD-PCR have shown excellent potential to analysis of ge- sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). netic structure of germplasm for several varieties of organisms Muscles from the five geckos were taken and stored at (Studer, 1992). Sodium dodecyl sulfate–polyacrylamide gel À20 °C. Muscles were grounded with 1 ml of 1· extraction electrophoresis (SDS–PAGE) is the most inexpensive, simple buffer; (10% SDS, 10 ml glycerol, 1 M Tris–HCl and 0.25 M and important tool that is widely used to differentiate the evo- EDTA, pH 8.8) and left overnight in refrigerator. The samples lution among the species (Ferguson, 1980; Avise, 1994; Mishra were centrifuged and the clear supernatants containing water- et al., 2010). The RAPD-PCR analysis is used to differentiate soluble proteins were used for electrophoresis. Muscle soluble the closely related species in order to determine the genetic protein fractions were separated exclusively on a vertical slab diversity between and within the species and is a useful method gel (19.8 cm · 26.8 cm · 0.3 cm) using the gel electrophoretic for showing of breeding populations (Welsh and Mc Clelland, apparatus (manufactured by APPEX) according to the method 1990; Williams et al., 1990, 1993). of Laemmli (1970). The final monomer concentration in the The present study was targeted to assess the genetic diver- 0.75 mm-thick slab gels was 12% (w/v) for the separating gel sity among five gekkonid species by using biochemical (pro- and 4% (w/v) for the stacking gel. Prior to loading, all samples tein) and molecular (RAPD-PCR) markers. were incubated in the presence of 1% (w/v) SDS and 100 mM Table 1 Scientific name, common name and localities of five gekkonid species from Egypt. Scientific name Common name Locality Tropiocolotes steudneri (Peters, 1869) Steudner’s Gecko, Steudner’s Pigmy Gecko Sinai Tropiocolotes nattereri (Steindachner, 1901) Natterer’s Gecko, BorsTaht El Hagar Sinai Tropiocolotes tripolitanus (Peters, 1880) Tripoli Gecko, Tripoli pigmy Gecko, BorsTaht El Hagar Sinai Tarentola mauritanica (Linnaeus, 1758) Moorish Gecko, Moorish wall Gecko, Crocodile Gecko Abu Rawash Tarentola annularis (Geoffroy, 1809) Egyptian Gecko, white spotted Gecko, Bors Abu ArbaNoqat Abu Rawash 186 N.H.M. Sayed Table 2 Binary data obtained for absence (0) and presence (1) from protein gel electrophoresis among five gekkonid species. BN RF MW Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 BF P 1 0.052 95 001000.2Unique 2 0.071 90 110000.4Polymorphic 3 0.108 83 111000.6Polymorphic 4 0.221 63 111111Monomorphic 5 0.247 59 010000.2Unique 6 0.260 57 000110.4Polymorphic 7 0.303 51 111111Monomorphic 8 0.334 48 111111Monomorphic 9 0.370 44 110000.4Polymorphic 10 0.394 41 000110.4Polymorphic 11 0.427 38 110000.4Polymorphic 12 0.451 36 111111Monomorphic 13 0.476 34 111000.6Polymorphic 14 0.522 30 110100.6Polymorphic 15 0.704 19 111111Monomorphic 16 0.739 18 000110.4Polymorphic 17 0.773 16 111111Monomorphic BN, band number; RF, relative front; MW, molecular weight in kilo Daltons (kDa); BF, band frequency; P, polymorphism. 1, Tropiocolotes steudneri;2,Tropiocolotes nattereri;3,Tropiocolotes tripolitanus;4,Tarentola mauritanica;5,Tarentola annularis. et al. (1989) with slight modifications. DNA quality and con- Table 3 Total number of bands, polymorphic bands, % of centration were determined by spectrophotometric analysis polymorphic bands, mean band frequency, unique bands of five and run in 0.7% agarose gel. Each sample of DNA was exam- gekkonid species for water soluble protein. ined by optical density values at 260 and 280 nm. Optical den- Monomorphic bands 6 sity ratios were evaluated and only good quality DNA samples Polymorphic (without unique) 10 were used in PCR. Unique bands 2 Polymorphic (with unique) 11 RAPD-PCR Total number of bands 17 Polymorphism (%) 64.70% Three primers from Kits OP-D, (Operon Technologies, Ala- Mean of band frequency 0.624 meda, CA, USA) were used for RAPD-PCR analysis (OPD- 02, OPD-03 and OPD-05). Only one primer (OPD-05) with se- quences D-05 50-TGAGCGGACA-30 showed some variations among five gekkonid species.